Noise induced hearing loss (NIHL) is the most common occupational disease for which no clinically effective drug is available. Since NIHL involves oxidation-linked loss of sensory hair cells and auditory neurons in the cochlea, there is a need for potent and effective antioxidants that offer protection to these tissues. In preliminary studies we have observed that the orally administered multifunctional antioxidant HK-2 that uses the innovative therapeutic strategy of combining both metal attenuating properties with free radical scavenging activities into one molecule, provides promising preventive protection against NIHL in rats. This proposal seeks to confirm, clarify, and expand on this observation by conducting in vivo dose-response studies in rats to determine the optimal dose of HK-2 required for NIHL protection. Subsequently, intervention studies will be conducted to determine whether the optimal dose of HK-2 is only protective when administered prior to damaging noise exposure or whether protection can also be obtained through intervention when administered at the onset of damaging noise exposure or even hours after exposure. To clarify the mechanism of action of HK-2, in vitro studies will be conducted in which are exposed to reactive oxygen species (ROS) which include superoxide, hydrogen peroxide and hydroxyl radicals, as well as reactive nitrogen species (RNS) derived from nitric oxide. HK-2, its monofunctional analogs, and parent compound will be used to define the relative importance of metal chelation versus free radical scavenging activity, the importance of mitochondrial function protection in these cochlear cells in the treatment of NIHL, and to demonstrate that HK-2 can protect against different sources of oxidative stress. The data obtained will provide the basis for an SBIR Phase 2 application to fund the necessary preclinical and toxicological studies required for obtaining a FDA investigational new drug (IND) application to develop multifunctional antioxidants for the clinical treatment of NIHL. House Ear Institute-Organ of Corti 1 (HEI-OC1) cells